Reactive oxygen species and free radicals cause deleterious effects in vivo. Antioxidant enzymes and low molecular-weight antioxidants protect organisms against these damaging species by removing or scavenging them. Mutation of these antioxidant enzymes or regulational disturbance of the low molecular-weight antioxidants causes genetic disorders, such as amyotrophic lateral sclerosis (ALS), or enhancement of deleterious effects. We investigated free radical generation in the glycation reaction, which crosslinks proteins found in diabetes mellitus, and the Cu,Zn-superoxide dismutase (SOD) mutant, which was found in familial ALS patients. The formation of alpha-dicarbonyl compounds from glucose seems to be an essential step for the crosslinking reaction, which leads to the formation of advanced glycation end-products (AGEs). To elucidate the mechanism for this reaction, we studied the reaction between a dicarbonyl compound, methylglyoxals, and L-alanines. The results showed that three types of free radical species were generated in this reaction: a crosslinked radical cation, the methylglyoxal dialkylimine radical cation; the methylglyoxal radical anion; and the superoxide radical. The radical site in protein crosslinks is stable and could be a reactive site for the generation of toxic superoxide anions over long duration in diabetes mellitus. Familial ALS patients have mutations of Cu,Zn-SOD. Transgenic mice that express a mutant SOD (G93A) were reported to develop ALS symptoms. To elucidate this effect, we cloned wild-type and the mutant cDNA, expressed proteins in insect cells (sf9), purified proteins, and determined the active-site Cu ion contents as well as their dismutation activities. Both enzymes have one Cu ion per subunit, and have identical dismutation activity. These results indicate that the ALS symptoms observed in G93A transgenic mice are not caused by the reduction of SOD activity, but rather caused by a gain-of-function, most likely by the enhancement of the free radical generating function, which we reported previously. We are currently preparing a large amount of purified proteins to compare the free radical generating activity of the mutant and wild-type human Cu,Zn-SOD.